Live animal imaging was performed as previously described 7 (link), 33 (link), 56 (link). Briefly, a Sutter Movable Objective Microscope (MOM) equipped with a pulsed femtosecond Ti:Sapphire laser (Chameleon Ultra II, Coherent) with two fluorescence detection channels was used for imaging (dichroic beamsplitter: FF520-Di02 (Semrock); blue emission filter: FF01-452/45 (Semrock); green emission filter: ET525/70M (Chroma); photomultiplier tubes: H7422-40 GaAsP (Hamamatsu)). Laser excitation wavelength was set to 830nm. Average laser power was <10-15mW at the tissue surface and adjusted with depth as needed to compensate for signal loss due to scattering and absorption. An Olympus 20× 1.0-NA water immersion objective was used for light delivery and collection. Z-stacks included up to 350 images, acquired at 1μm axial step size, used a 2-frame average, 512 × 512 pixel resolution, and 2.0x-10x zoom (corresponding to 350μm-72μm fields of view). Time-lapse recordings typically included 60–70 images/stack, acquired at 1.0–1.2 μm axial step size, used a 2-frame average, 60 stack repeats (corresponding to approximately 94 min total recording duration), 512 × 512 pixel resolution, and 3.3x-5x zoom (corresponding to 212 μm - 142 μm fields of view). Up to thirteen z-stacks, and four to seven time-lapse recordings were acquired per animal in layers 1 and 2 of the somatosensory cortex.